Western Blotting of Cellular Proteins

Western blotting was performed strictly according to previously described procedures [19 (link)]. Briefly, cells were washed with ice-cold PBS and soluble proteins were extracted with cell lysis buffer (100 mM Tris-HCl (pH = 8), 150 mM NaCl, 1% NP-40, phosphatase and protease inhibitor cocktail tablets (Abcam, Shanghai, China)) according to the manufacturer’s protocol. The protein concentration of each sample was determined by a BCA Kit (Beyotime, China). An equal amount of protein was separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Millipore). The membrane was then blocked with 5% non-fat milk for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. Antibodies were obtained from Sigma-Aldrich (HA, # H9658) and Proteintech (Wuhan, China) (β-actin, # 60008-1-Ig). Signals were visualized using infrared imaging systems and quantified using ImageJ software v 1.0.20 (Odyssey CLX, LiCor Biosciences, Lincoln, NE, USA).